life
Review
Cell Immortality: In Vitro Effective Techniques to Achieve and
Investigate Its Applications and Challenges
Mahla Chalak 1,2,† , Mahdi Hesaraki 2,3,† , Seyedeh Nasim Mirbahari 1,4 , Meghdad Yeganeh 5 , Shaghayegh Abdi 2 ,
Sarah Rajabi 6, * and Farhid Hemmatzadeh 7, *
Citation: Chalak, M.; Hesaraki, M.;
Mirbahari, S.N.; Yeganeh, M.; Abdi, S.;
Rajabi, S.; Hemmatzadeh, F. Cell
Immortality: In Vitro Effective
Techniques to Achieve and Investigate
Its Applications and Challenges. Life
2024, 14, 417. https://doi.org/
10.3390/life14030417
Academic Editor: Muhammad Rafehi
Received: 28 February 2024
Revised: 16 March 2024
Accepted: 17 March 2024
Published: 21 March 2024
Copyright: © 2024 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Life 2024, 14, 417. https://doi.org/10.3390/life14030417
1 Department of Developmental Biology, School of Basic Sciences and Advanced Technologies in Biology,
University of Science and Culture, Tehran P.O. Box 871-13145, Iran
2 Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem
Cell Biology and Technology, ACECR, Tehran 1665659911, Iran
3 Department of Photo Healing and Regeneration, Medical Laser Research Center, Yara Institute, ACECR,
Tehran 1417613181, Iran
4 Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive
Biomedicine, ACECR, Tehran 1665659911, Iran
5 School of Biotechnology, College of Science, University of Tehran, Tehran 1417935840, Iran
6 Department of Cell Engineering, Cell Science Research Center, Royan Institute for Stem Cell Biology and
Technology, ACECR, Tehran P.O. Box 16635-148, Iran
7 Australian Centre for Antimicrobial Resistance Ecology, School of Animal & Veterinary Sciences, University of
Adelaide, Roseworthy Campus, Roseworthy, SA 5371, Australia
* Correspondence: srajabi@royaninstitute.org (S.R.); farhid.hemmatzadeh@adelaide.edu.au (F.H.);
Tel.: +98-(21)-23562506 (S.R.); +61-8-8313-7723 (F.H.); Fax: +98-(21)-22310406 (S.R.); +61-8-8313-7956 (F.H.)
† These authors contributed equally to this work.
Abstract: Cells are very important to researchers due to their use in various biological studies
in in vitro and in vivo settings. This importance stems from the short lifespan of most cells under
laboratory conditions, which can pose significant challenges, such as the difficulties associated with
extraction from the source tissue, ethical concerns about separating cells from human or animal
models, limited cell passage ability, and variation in results due to differences in the source of the
obtained cells, among other issues. In general, cells in laboratory conditions can divide into a limited
number, known as the Hayflick limit, due to telomere erosion at the end of each cellular cycle. Given
this problem, researchers require cell lines that do not enter the senescence phase after a limited
number of divisions. This can allow for more stable studies over time, prevent the laborious work
associated with cell separation and repeated cultivation, and save time and money in research projects.
The aim of this review is to summarize the function and effect of immortalization techniques, various
methods, their advantages and disadvantages, and ultimately the application of immortalization and
cell line production in various research fields.
Keywords: immortalization; cell division; telomeres; hTERT; cell line
1. Introduction
In 1965, Leonard Hayflick introduced the idea that cells possess a mechanism for
keeping track of the number of times they divide, a concept later termed the Hayflick
limit, which aligns with the number of replication cycles [1]. The molecular basis of
this phenomenon is based on the gradual shortening of telomeric DNA (Figure 1). The
shortening of telomeres occurs with each cell division, ultimately causing cells to reach their
Hayflick limit, whereby growth stops after approximately 60 times population doubling [2–5].
When telomeres become too short and cannot function naturally, cellular senescence in
the M1 phase (a cellular growth arrest, also called the M1 stage) occurs [5–7]. Thus, very
short telomeres are identified by cells as double-stranded breaks and create DNA damage
responses that include cellular apoptosis and replicative senescence mechanisms [4].
https://www.mdpi.com/journal/life
Life 2024, 14, x FOR PEER REVIEW 2 of 23

in the M1 phase (a cellular growth arrest, also called the M1 stage) occurs [5–7]. Thus, very
Life 2024, 14, 417 short telomeres are identified by cells as double-stranded breaks and create DNA damage2 of 22
responses that include cellular apoptosis and replicative senescence mechanisms [4].

Figure 1. The Hayflick limit. Naturally, with each cell division, the telomeres at the ends of chromo-
Figure chromo-
somes become
somes become shorter,
shorter, aa phenomenon
phenomenon known
known asas the
the Hayflick
Hayflick limit.
limit. The
The final
final stage
stage is
is also
also known
known as
as
cellular aging.
cellular aging.

Therefore, due to the the short

short lifespan
lifespan of of cells
cells inin laboratory
laboratory conditions,
conditions, aa continuous
continuous
supply
supply of these cells from their specialized sources seems necessary for scientific scientific research.
research.
However,
However,the therepeated
repeatedprocurement
procurementofofcells cellsrequires
requires much
much skill and
skill andexperience
experience if the donor
if the do-
is
noravailable, as mature
is available, cellscells
as mature fromfrom
theirtheir
origin tissues
origin (such(such
tissues as vascular tissue)
as vascular are difficult
tissue) are dif-
to obtain
ficult [8]. [8].
to obtain
Furthermore,
Furthermore, the the preparation
preparation of of primary
primary human human and and animal
animal tissues
tissues for
for the
the extraction
extraction
and
and isolation of various cell types is fraught with significant challenges, including the the
isolation of various cell types is fraught with significant challenges, including po-
potentially invasive
tentially invasive nature
nature ofofthe
theprocess,
process,an anincreased
increasedrisk riskofofinfection,
infection,pain,
pain, ethical
ethical issues,
issues,
and the difficulty
and the difficulty ofofthe
theprocess
processitself.
itself.InInsome
some cases,
cases, somesome patients
patients maymaynotnot consent
consent to
to the
the
use use of excess
of excess samples
samples separated
separated from from
their their organs,
organs, which which
cancan delay
delay access
access to cellular
to cellular re-
resources in research. In addition to all these problems, obtaining
sources in research. In addition to all these problems, obtaining cells from various sources cells from various
sources
and their and their availability
availability for conducting
for conducting a studyacan study
leadcan tolead to errors
errors and changes
and changes in the in the
inter-
interpretation and results of research. For example, as human
pretation and results of research. For example, as human umbilical vein endothelial cellsumbilical vein endothelial
cells
(HUVECs)(HUVECs) are obtained
are obtained from the from the umbilical
umbilical cord, theycord,arethey are not
not only only influenced
influenced by maternal by
maternal hormones but also affected by embryonic gonads.
hormones but also affected by embryonic gonads. In other words, even the fetal sex fromIn other words, even the fetal
sex
whichfrom which HUVEC
HUVEC cells are cells are isolated
isolated can playcan play a significant
a significant role in role in the reproducibility
the reproducibility of the
of the results. In fact, various studies have shown sexual differences in the umbilical cord
results. In fact, various studies have shown sexual differences in the umbilical cord be-
between men and women in terms of gene expression, protein expression, cell survival,
tween men and women in terms of gene expression, protein expression, cell survival, tu-
tubule formation capacity, autophagy, cellular ATP and metabolite levels, oxidative stress,
bule formation capacity, autophagy, cellular ATP and metabolite levels, oxidative stress,
and angiogenesis [9–12]. This challenge can also apply to other cells. Therefore, researchers
and angiogenesis [9–12]. This challenge can also apply to other cells. Therefore, research-
need immortalized cells that do not enter the senescence phase after several cell divisions,
ers need immortalized cells that do not enter the senescence phase after several cell divi-
considering the limitations of obtaining more adaptable cells over time.
sions, considering the limitations of obtaining more adaptable cells over time.
Immortalized cell lines are modified cells that can be grown indefinitely [13]. Ideally,
Immortalized cell lines are modified cells that can be grown indefinitely [13]. Ideally,
immortalized cells are genetically and phenotypically similar or identical to their source tis-
immortalized cells are genetically and phenotypically similar or identical to their source
sue and can reproduce and be cultured in the long term. Immortalized cell lines can be used
tissue and can reproduce and be cultured in the long term. Immortalized cell lines can be
in most research instead of primary cells because they offer several advantages, including
used in most research instead of primary cells because they offer several advantages, in-
cost-effectiveness, ease of use, unlimited availability of materials, and the bypassing of
cludingconcerns
ethical cost-effectiveness,
associated easewithof use, unlimited
animal and human availability
tissue useof[14–16].
materials, and the bypass-
ing of ethical concerns associated with animal and human
In the late 1950s, George Otto Gey, head of tissue culture research tissue use [14–16].
at Johns Hopkins,
initiated the process of immortalization and established a non-aging cellatline
In the late 1950s, George Otto Gey, head of tissue culture research Johns
calledHopkins,
HeLa,
initiated the process of immortalization and established a
derived from a rare adenocarcinoma found in the cervix of a young woman named Hen- non-aging cell line called HeLa,
rietta Lacks. This marked the first instance of such a phenomenon [17,18]. Prior to this,
cells derived from other human cells could only survive for a few days in culture, but
the behavior of Lacks’s tumor cells was different. This was the first successful attempt to
Life 2024, 14, 417 3 of 22

immortalize and maintain human cells in vitro because the ability of Hela cells made them
useful in various biological studies [19–23].
In fact, HeLa cells have an active version of telomerase during cell division [24], which
copies telomeres repeatedly. This prevents the gradual shortening of telomeres, which
plays a role in cell aging and, ultimately, cell death. Thus, cells evade the Hayflick limit,
resulting in unlimited cell division and immortality. Major discoveries have been made
using this cell line, including the development of the polio vaccine in 1953 [25], the link
between human papillomavirus (HPV) and cervical cancer, and the role of telomerase in
chromosome maintenance [26].
Generally, various cell lines have revolutionized scientific investigations and have
found applications in vaccine development, drug testing for metabolism and toxicity,
generating antibodies, exploring gene functions, creating artificial tissues like synthetic
skin, and producing biologic substances such as therapeutic proteins [16,19,26]. The aim of
this review is to address the process of immortalization of cells and various methods of
creating different cell lines and their use in research.

2. The Application of Immortalized Cell Lines

Immortalized cell lines are a powerful tool for biological, biochemical, and biological
growth, differentiation, and aging studies. They are also used in immunology, hematology,
cancer biology, and toxicology research. Additionally, for therapeutic purposes, studying
immortalized cells will be useful in achieving better results for regenerative medicine [27].
Cell line immortalization can also help cell biologists achieve their goals of treating diseases
and improving health factors. For example, they have used immortalized cell lines as model
systems for studying neuronal development and performance recovery in neurological
disease models, such as Huntington’s disease [28,29].
In general, the use of these cell lines can be useful for clinical development related
to the treatment of Huntington’s disease patients [29]. Another example is human vo-
cal fold epithelial cells, which are a valuable tool for studying epithelial–fibroblast cell
interactions that dictate the disease and health of this specialized tissue [30]. The RPE-1
human retinal pigment epithelial cell line has also been widely used to study physiological
events in human cellular culture systems [31]. Pig endothelial cells can be also used as a
laboratory model to study the properties of the blood–brain barrier and some hemorrhagic
diseases [32]. Encapsulated cell technology is also a useful approach for the continuous and
local delivery of genetically modified therapeutic proteins. However, the clinical develop-
ment of encapsulated cell technology to deliver therapeutic proteins from macro-capsules
is still limited due to the lack of a compatible allogeneic cell line for therapeutic purposes
in humans [33].
In this regard, it is possible to create appropriate immortal cell lines, such as the human
myoblast cell line created in the study by Lathuiliere A. and colleagues. Valuable results
have also been achieved using enclosed cell technology. As an example, non-immortalized
human lung epithelial cells created by hTERT overexpression without the use of viral
oncogenes have been used to investigate various aspects of lung cancer, such as epithelial-
to-mesenchymal transition and the cancer stem cell theory. The use of non-immortalized
lung epithelial cells has improved researchers’ understanding of lung cancer pathogenesis,
and these models can be valuable research tools [34]. Additionally, creating cell lines that
preserve genetic information and drug responses can enable more drug screening and
mechanistic studies [35].
The immortalized NTera2 (NT2) cell line has undergone a thorough examination for
its potential in brain grafting. Notably, these cells have been safely employed in clinical
trials focused on treating brain injuries [36,37]. Lately, a method involving the lentiviral
gene transfection of c-MYC has been employed to create immortalized megakaryocyte cell
lines, regulated by the TET-on system, using blood cell precursor cells derived from iPSCs
(induced pluripotent stem cells). These cells have the capability to produce platelets for
use in clinical trials [38]. Cell lines that are not immortalized can serve as valuable tools for
 gene transfection of c-MYC has been employed to create immortalized megakaryocyte cell
lines, regulated by the TET-on system, using blood cell precursor cells derived from iPSCs
(induced pluripotent stem cells). These cells have the capability to produce platelets for
Life 2024, 14, 417 use in clinical trials [38]. Cell lines that are not immortalized can serve as valuable 4 of 22 tools
for investigating the effectiveness and potential harm of drugs. For instance, human cell
lines have been extensively employed in pharmacogenomic research concerning cancer,
aiding in the
investigating theforecast of clinical
effectiveness responses,
and potential harm contributing to the development
of drugs. For instance, human cell lines of phar-
have been extensively
macogenomic theoriesemployed in pharmacogenomic
for subsequent experiments,research concerning
and uncovering cancer,
fresh aidinginto the
insights
invarious
the forecast
factorsof clinical responses,
influencing drug contributing
responses. Within to the development
the category of ofpharmacogenomic
model system cell lines,
theories for subsequent experiments, and uncovering
immortalized types like EBV-transformed lymphoblastoid cell lines fresh insights into the(LCLs)
variousare
factors
frequently
influencing drug responses. Within the category of model system cell lines,
utilized to assess how genetic diversity impacts the effectiveness and safety of drugs [39]. immortalized
types like EBV-transformed lymphoblastoid cell lines (LCLs) are frequently utilized to
Additionally, if we aim to apply this technique in a different research area, it would
assess how genetic diversity impacts the effectiveness and safety of drugs [39].
pertain to the creation of immortalized cell lines for food production, a significant hurdle
Additionally, if we aim to apply this technique in a different research area, it would
in the field
pertain to theof cell agriculture.
creation This field
of immortalized is dedicated
cell lines to generating
for food production, meat andhurdle
a significant otherinanimal-
derived products via tissue engineering and synthetic biology. Specifically,
the field of cell agriculture. This field is dedicated to generating meat and other animal- these cultured
meat cell
derived lines must
products meetengineering
via tissue criteria forand food safety,biology.
synthetic be capable of large-scale
Specifically, propagation
these cultured
and cell
meat differentiation
lines must meet in an efficient
criteria manner,
for food safety,andbe exhibit
capable taste, texture,propagation
of large-scale and nutritional
and qual-
ities that appeal
differentiation to efficient
in an consumers [40]. and exhibit taste, texture, and nutritional qualities
manner,
that appeal
In thistocontext,
consumers [40]. primary cells for cultured meat production necessitates the
utilizing
In this context,
maintenance utilizing
of donor primary
animal herds cells
forfor cultured
sample meat production
preparation, necessitates
with regular the
and approved
maintenance of donor animal herds for sample preparation, with regular
sample collection for food production. In contrast to primary cell cultures, immortalized and approved
sample collection
cell lines for food production.
do not undergo senescenceIn andcontrast to primary
can undergo cell cultures,
limitless divisions.immortalized
As a result, they
cell lines do not undergo senescence and can undergo limitless divisions. As a result, they
are more straightforward to investigate and enable the production of cultured meats that
are more straightforward to investigate and enable the production of cultured meats that
are both safer and more consistently achieved (Figure 2), eliminating the requirement for
are both safer and more consistently achieved (Figure 2), eliminating the requirement for
animalbiopsies
animal biopsies [27].
[27].

Figure 2. A look at some of the applications of immortalized cells in different types of research.
Figure 2. A look at some of the applications of immortalized cells in different types of research.
3. Telomeres
3. Telomeres
The important role of telomeres in ensuring chromosomal stability was first proposed
in the The
1930simportant
by Barbara McClintock
role [41],in
of telomeres who workedchromosomal
ensuring on corn, and Hermann Muller,
stability was firstwho
proposed
worked on fruit flies [42]. Both researchers suggested that the ends of chromosomes
in the 1930s by Barbara McClintock [41], who worked on corn, and Hermann Muller, who have
special
worked structures that are
on fruit flies [42].necessary for chromosomal
Both researchers suggestedstability.
that theMuller coined
ends of the term have
chromosomes
telomere from the Greek words telos, meaning “end”, and meros, meaning
special structures that are necessary for chromosomal stability. Muller coined “part”. Telom-
the term
eres are necessary for protecting the ends of chromosomes, creating chromosomal stability,
telomere from the Greek words telos, meaning “end”, and meros, meaning “part”. Telo-
and ensuring the separation of genetic material into daughter cells after cell division [43,44].
meres are necessary for protecting the ends of chromosomes, creating chromosomal sta-
Telomeres are nuclear protein complexes located at the ends of eukaryotic cell chro-
bility, and
mosomes ensuring
[45]. the separation
This arrangement of genetic
inhibits material into
the identification daughter
of linear cells afterends
chromosome cellas
division
[43,44].
double-stranded DNA breaks [46]. The DNA sequence of telomeres is characterized by
repeated tandem sequences (TTAGGG) in all vertebrates (Figure 3) [47].
 Telomeres are nuclear protein complexes located at the ends of eukaryotic cell chro-
mosomes [45]. This arrangement inhibits the identification of linear chromosome ends as
Life 2024, 14, 417 double-stranded DNA breaks [46]. The DNA sequence of telomeres is characterized 5by of 22
repeated tandem sequences (TTAGGG) in all vertebrates (Figure 3) [47].
Telomeric DNA typically ends with a G-rich overhang, which consists of unpaired
nucleotides atTelomeric
the 3’ endDNAof a DNA molecule,
typically ends with ranging between
a G-rich 50 and
overhang, 300consists
which nucleotides [48].
of unpaired
The lengths of theseatrepeats
nucleotides vary
the 3’ end of between chromosomes
a DNA molecule, rangingand species
between (Figure
50 and 3) [48–50]. [48].
300 nucleotides
The lengths
In humans andofmice,
thesethe
repeats vary
length ofbetween
telomere chromosomes andends
repeats at the species
of (Figure 3) [48–50].
individual chro-
In humans and mice, the length of telomere repeats at the ends
mosomes varies significantly among cells. Human chromosome ends usually have detect- of individual chromo-
somes varies significantly among cells. Human chromosome ends usually
able telomere repeats between 0.5 and 15 kilobases (kb), depending on factors such as have detectable
telomere repeats between 0.5 and 15 kilobases (kb), depending on factors such as tissue
tissue type, donor age, and cell replication history. Specific changes in telomere length
type, donor age, and cell replication history. Specific changes in telomere length occur at
occur at the ends of individual human chromosomes, with the average length varying
the ends of individual human chromosomes, with the average length varying between
between chromosome
chromosomeends. ends.For
Forinstance,
instance,chromosome
chromosome17p 17ptypically
typically has
has shorter
shorter telomeres
telomeres than
than other chromosome ends [51,52].
other chromosome ends [51,52].

Figure 3. Telomerase
Figure 3. Telomerase components components
and TERTand TERT structure.
structure. Telomerase Telomerase
is composed is composed of three
of three main main
com-
components—hTERT,
ponents—hTERT, hTERC, andhTERC,DKC1.and The DKC1.
hTERC TheishTERC is associated
associated with DKC1
with DKC1 and and
smallsmall nucleolar
nucleolar
RNPs,NHP2,
RNPs, NOP10, NOP10,and NHP2, and The
GAR1. GAR1. The sequence,
sequence, by adding
by adding repetitive
repetitive TTAGGG TTAGGG sequences
sequences to the
to the
end of theend
telomere, preventsprevents
of the telomere, its shortening and naturally
its shortening prevents
and naturally cell aging.
prevents The The
cell aging. protein complex
protein complex
shelterin, shelterin,
or telosome, protects protects
or telosome, telomere ends. The
telomere shelterin
ends. complex
The shelterin is made
complex up ofuptelomeric
is made repeat
of telomeric repeat
binding factors
binding1 and 2 (TERF1
factors 1 and 2and TERF2),
(TERF1 repressor/activator
and TERF2), proteinprotein
repressor/activator 1 (RAP1), protection
1 (RAP1), of te- of
protection
lomeres 1 telomeres
(POT1), TERF1 interacting
1 (POT1), nuclear factor
TERF1 interacting nuclear2 factor
(TINF2), and TPP1.
2 (TINF2), and TPP1.

In human In nuclear
human nuclear cells as
cells such such as immune
immune system
system cells,
cells, the theaverage
averagetelomere
telomere length
lengthde-
creases significantly with age. The shortening of telomeres is a
decreases significantly with age. The shortening of telomeres is a presumed tumor sup- presumed tumor suppressor
pathway mediated, in part, by the activation of cellular aging signals. The first scientist
pressor pathway mediated, in part, by the activation of cellular aging signals. The first
to link telomeres with planned cell division was Alexei Olovnikov [53]. He proposed
scientist to link telomeres with planned cell division was Alexei Olovnikov [53]. He pro-
that human somatic cells may not be able to correct chromosomal shortening that occurs
posed thatduring cellsomatic
human cellsHe
replication. may not be able
suggested that to correctnucleotide
repeated chromosomal shortening
sequences that
in telomeres
occurs during cellasreplication.
could act He suggested
a buffer to protect downstream that repeated
genes nucleotide sequences
from chromosome shortening inineach
telo-cell
meres could act as a buffer to protect downstream genes from chromosome shortening
division. Additionally, he had significant insights and suggested that the length of repeated in
each cell sequences
division. Additionally,
could determine hethehad significant
number of DNA insights and suggested
replication cycles. that the length
Watson also
of repeated sequences recognized
could that the
determine thenature
number of unidirectional
of DNA replicationDNA replication
cycles. for complete
end-to-end
Watson telomere replication
also recognized poses of
that the nature a problem [54]. The
unidirectional DNAfirstreplication
direct observations
for com- of
telomeres’ direct association with aging in 1986 occurred when Cooke
plete end-to-end telomere replication poses a problem [54]. The first direct observations and Smith discovered
that the average length of telomere repeats covering sex chromosomes in sperm cells is
of telomeres’ direct association with aging in 1986 occurred when Cooke and Smith dis-
much greater than in adult cells [55]. They considered the possibility that adult cells may be
covered that the average length of telomere repeats covering sex chromosomes in sperm
deficient in the telomerase enzyme, which had recently been discovered in the single-cell
cells is much greater
organism than in adult
Tetrahymena [56]. cells [55]. They considered the possibility that adult
Life 2024, 14, 417 6 of 22

Several reports confirmed the decrease in average telomere length with cell division
in fibroblasts [6,57,58] and with age in somatic blood and colon cells [59], but not in germ
cells. These observations confirmed that somatic cells apparently cannot maintain telomere
length, but with the use of telomerase reactivation techniques, it can be preserved to
some extent.

4. Immortality and a Review of Its Techniques

Numerous biotechnological approaches have been employed to manipulate the cel-
lular genome in order to acquire immortalized cell lines. Nonetheless, the foremost and
extensively applied techniques for achieving this involve the introduction of viral onco-
proteins and telomerase reverse transcriptase (TERT) [60]. The immortality conferred by
viral oncoproteins is intricately linked to the deactivation of cell cycle-regulating proteins
(such as p16, p14, p21, p53, and Rb). Through this pathway, viral oncoproteins can induce
the deactivation of tumor suppression mechanisms and may even trigger the expression
of telomerase [61].
Furthermore, a correlation exists between the deactivation of pRb, cellular aneuploidy,
and chromosomal instability. As a result, pRb and p53 emerge as pivotal proteins governing
cellular senescence and replication. During senescence, an active, hyperphosphorylated
form of pRb is present. This form binds with members of the E2F protein family, leading to
the deactivation of certain genes involved in the G1/S phase transition. It is worth noting
that Rb’s growth-inhibiting function appears to operate independently of p53. In human
cells, p53 can initiate senescence autonomously without relying on Rb [62]. However, in
general, the techniques used for cell immortalization by inactivating the two main tumor
suppressor pathways, pRb and p53, can lead to genomic instability and the formation of
polyploidy and altered chromosome numbers. Losing the function of the p53 gene can
cause genomic instability and thus disrupt the ability of the genome to replicate accurately.
This has been observed in about half of human malignant cancers [63] and underscores
the vital role of p53 in tumor suppression. Therefore, the use of these techniques cannot be
considered a safe and risk-free option for cell immortalization.

4.1. Viral Genes Can Control the Cell Cycle

As viruses rely on the ability to replicate within living organisms to survive, they are
able to manipulate or accelerate the cellular cycle for their own benefit [64]. One of the
methods to achieve this is through targeting the expression of Rb and p53 proteins. Viral
oncogenes are also able to disable pRb and p53, thus overcoming the M1 barriers, which
inhibit the growth and replication of natural cells, and significantly increasing the lifespan
of cells [65].

4.1.1. Simian Virus 40

The Simian Virus 40 (SV40) encodes two proteins, Large T antigen (LT) and Small
T antigen (ST), which help induce virus-associated tumors. SV40 T antigen is currently
used for cell transfection in different types of cells and can generate immortal cell lines
by binding and disabling p53 and Rb proteins [66]. Both of these proteins have evolved
to specifically target crucial cellular regulators and modify their functions. The Large T
antigen, for instance, targets various known proteins encompassing 708 amino acids. These
include three members of the retinoblastoma protein family (pRb, p107, and p130), as well
as members of the Cap-binding protein (CBP) adaptor protein family, such as p300 and
p400, along with the tumor suppressor protein p53. On the other hand, the Small T antigen
affects the activity of the pp2A phosphatase and activates the cyclin A promoter. Notably,
the LT protein plays a central role in conferring SV40’s extended lifespan, primarily due
to its capability to interact with growth suppressors like pRb and p53 (Figure 4) [67] and
suppress the p53 pathway [68].
Life 2024, 14, x FOR PEER REVIEW 7 of 23

Life 2024, 14, 417 to its capability to interact with growth suppressors like pRb and p53 (Figure 4) [67] and
7 of 22
suppress the p53 pathway [68].

Anoverview
Figure4.4.An
Figure overviewofof various
various types
types of of
cellcell immortalization
immortalization methods.
methods.

Amongthe
Among thecell
celltypes
typesthat
that achieve
achieve immortalization
immortalization through
through this
this mechanism,
mechanism, human
human
proximal epithelial cells serve as an example, retaining their differentiation properties[69].
proximal epithelial cells serve as an example, retaining their differentiation properties
Conversely,
[69]. Garcìa-Mesa
Conversely, Garcìa-Mesa and collaborators successfully
and collaborators immortalized
successfully microglial
immortalized cells for
microglial
the purpose of studying the latency and regulation of the human immunodeficiency
cells for the purpose of studying the latency and regulation of the human immunodefi- virus
ciency virus (HIV) in the central nervous system (CNS). Importantly, they managed tothe
(HIV) in the central nervous system (CNS). Importantly, they managed to preserve
majoritythe
preserve of majority
the primaryof theglial cell’sglial
primary phenotypic and functional
cell’s phenotypic characteristics
and functional [70]. Con-
characteristics
versely, pre-adipocytes showed aberrant differentiation after immortalization
[70]. Conversely, pre-adipocytes showed aberrant differentiation after immortalization with the
SV40 T antigen. In fact, the ability of the SV40 T antigen to block the transcription
with the SV40 T antigen. In fact, the ability of the SV40 T antigen to block the transcription factor
p300/cAMP-response element-binding protein (CBP), which is essential
factor p300/cAMP-response element-binding protein (CBP), which is essential for adipo- for adipocyte
differentiation,
cyte inhibits
differentiation, pre-adipocyte
inhibits pre-adipocyte differentiation [71].
differentiation [71].
4.1.2. Human Papillomavirus (HPV)
4.1.2. Human Papillomavirus (HPV)
HPV is a small, double-stranded DNA virus that infects mucosal and cutaneous
HPV is a small, double-stranded DNA virus that infects mucosal and cutaneous epi-
epithelial tissues [72]. High-risk strains, including HPV-8/16/18/31, cause malignant
thelial tissues [72]. High-risk strains, including HPV-8/16/18/31, cause malignant lesions,
lesions, while low-risk strains, including HPV-6/11, cause benign warts and lesions [73].
while low-risk strains, including HPV-6/11, cause benign warts and lesions [73]. The E6
The E6 and E7 proteins, encoded by high-risk strains such as HPV-16/18, are classified as
oncoproteins [74]. When used in immortalization, E6 activates telomerase and accelerates
the degradation of p53 by proteasome S26, while E7 can inactivate Rb by preventing the
binding between pRb and the transcription factor E2F [75,76]. For example, Trakarnsanga
and colleagues achieved the immortalization of early adult erythroblasts by employing
Life 2024, 14, 417 8 of 22

HPV-16 oncoproteins. This led to the creation of stable cells capable of producing red
blood cells [77].
Furthermore, by employing viral oncoproteins E6 and E7, along with the overexpres-
sion of the T SV40 antigen, epithelial cells located on the surface of the ovary have been
successfully immortalized [78]. Overall, the transfer of certain cell types with E6/E7 HPV
oncoproteins creates a cell line that retains many characteristics of primary tissue cells.

4.1.3. Human T-Lymphotropic Virus (HTLV)

HTLV is a human RNA retrovirus that causes leukemia and adult T-cell lymphoma.
There are two types, HTLV-1 and HTLV-2, which have the ability to infect lymphocytes un-
der laboratory conditions, although clinically, HTLV-2 is less pathogenic than HTLV-1 [79].
Types 1 and 2 encode for the Tax1 and Tax2 proteins, respectively, which are necessary for
the infection of human T-cells by the associated viruses. Due to the greater pathogenic
power of HTLV-1, the number of growth-induced cells by Tax2 was much greater than the
cells induced by Tax1, and the activity of Tax2 was far higher than Tax1 [80].

4.1.4. Adenoviruses
Adenoviruses are common DNA viruses found in animals and humans and are
often observed in adults and children [81]. The 12S E1A gene product from adenovirus,
belonging to the oncoprotein class, has the capability to establish primary cells as cell
lines. It is encoded by two exons. Extensive mutational studies have revealed that four
specific regions of the 12S E1A gene, derived from both exons, play a critical role in
prolonging the lifespan of primary epithelial cells. While the expression of two of these
regions is essential for activating quiescent cells and initiating the cell cycle, it alone cannot
confer immortality or extend their lifespan in culture. These two regions are encoded
by exon 1. The third region within exon 1, whose function remains unidentified, is also
indispensable for this process. Moreover, these three regions are crucial for cooperating
with 12S and an activated ras gene in triggering tumor formation. The fourth region is
necessary for sustaining the proliferation of cells, prolonging their lifespan in culture, and
prompting autocrine growth factor production. Cells immortalized by both wild-type
12S and its mutant variants maintain their epithelial characteristics, and they continue to
express intermediate filament proteins like keratin and vimentin [82]. For example, by
transferring retroviral vectors containing coding sequences of 12S or 13S E1A, they caused
the proliferation and immortalization of epithelial cells in primary cultures of the kidneys,
liver, heart, pancreas, and thyroid of mice [83].

4.1.5. Epstein–Barr Virus (EBV)

EBV is a double-stranded DNA virus that infects B lymphocytes. Immortalizing B
cells is an effective method for inducing the long-term growth of some human B cells in
laboratory conditions [84]. In fact, this virus can immortalize cells and convert them into
lymphoblastoid cell lines that carry EBV [85]. These cells effectively induce specific T cell
responses against EBV in laboratory conditions due to the presentation of viral antigens [86].
It has been shown that EBNA-2 is genetically essential for B cell immortalization by EBV.
Experiments have shown that EBNA-2 affects the accumulation of viral and cellular RNAs,
and LMP may also be necessary for immortalization as it can affect the growth properties
of human lymphoid and epithelial cells. EBNA-1 may also be necessary for the immortal-
ization of a B cell for EBV, as it appears to be necessary for the maintenance of viral DNA
replication in the replicating cell population [87].
According to these results, we suggest that using viral genes for immortalization may
affect the genome of target cells that can produce active oncogenes. So, this process is not
recommended for the immortalization of primary cells.
Life 2024, 14, 417 9 of 22

4.2. The Overexpression of Specific Genes for Immortalization

4.2.1. The HOX Gene Family and Lhx2
The overexpression of the HOX11 and TLX1 genes can lead the hematopoietic pre-
cursor cells, mouse fetal liver, or bone marrow towards immortalization. A wide range of
hematopoietic cell types have been immortalized using these methods, including erythroid,
megakaryocytic, monocytic, myelocytic, and multipotent cells [88]. Studies have also shown
that myelomonocytic, megakaryocytic, and mast cell progenitor immortalization in mice
can be achieved using the Hox-2.4 gene [89]. It has also been reported that the expression
of the HOXa9 gene can immortalize a promyelocyte. In fact, Hoxa9 expression in primary
murine marrows can immortalize a delayed myelomonocytic progenitor and prevent final
differentiation into granulocytes or monocytes in the presence of granulocyte–macrophage
colony-stimulating factor or interleukin-3 [90]. The expression of the Lhx2 gene in mature
bone marrow-derived hematopoietic stem/progenitor cells can provide the possibility
of producing hematopoietic stem/progenitor cells dependent on sustained growth fac-
tor, which can produce erythroid, myeloid, and lymphoid cells after grafting into mice.
Therefore, Lhx2 is capable of immortalizing multipotent hematopoietic stem/progenitor
cells, which can create functional outcomes after grafting into treated hosts [91].

4.2.2. c-Myc Gene Expression

Another set of genes employed in the immortalization of cells belongs to the myc
family. This family encompasses a group of oncogenes: c-Myc, N-Myc, L-Myc, and B-Myc.
The expression of c-Myc is predominantly observed in actively proliferating cells, whereas
N-Myc and L-Myc play roles in differentiation processes. Among these oncogenes, the
c-Myc gene has been the subject of the most extensive studies [61]. It is worth noting that
there is an association between p53 and c-Myc, as the Myc signaling pathway governs
both apoptosis and cell immortalization, with the latter being contingent on the status
of p53 [92].
While the overexpression of c-Myc leads to DNA damage and promotes genomic
instability, it also circumvents the pro-apoptotic functions of p53 [93]. The excessive activity
of c-Myc contributes to apoptosis by involving NF-κB mediation. Thus, the malfunctioning
signaling of NF-κB is a necessary condition for Myc-induced carcinogenesis [94]. De Filippis
and colleagues devised a technique to immortalize neural stem cells (NSCs) by introducing
a retroviral vector carrying a mutated variant of c-Myc (c-Myc T58A) [95].
On the other hand, Li and colleagues also achieved a perpetual population of NSCs
through L-Myc transduction [96]. Myc also stabilizes telomere length in human prostate
epithelial cells (HPrECs) through the regulation of hTERT gene overexpression. Overall,
HPrECs that are immortalized by the c-Myc gene maintain many normal cell character-
istics, such as the induction of growth cessation in response to the Ras oncogene, intact
p53 response, and absence of gross karyotypic abnormalities. However, they lack an
Rb/p16INK4a surveillance checkpoint, which is a weakness of this method [97].

4.2.3. CDK4 Gene Expression

CDK4 expression, along with increased hTERT gene expression, has been used for the
immortalization of human bronchial epithelial cells [98]. Additionally, CDK4 expression,
along with cyclin D1 and increased telomerase activity, has been used for the immortaliza-
tion of human myogenic cells derived from healthy and diseased muscles [99].

4.2.4. TERT Gene Expression

As mentioned, the risk of oncogenic integration into chromosomes still raises vari-
ous safety concerns when using an oncogenic transferable agent in host cells [61]. Using
hTERT as a means of achieving immortalization through less phenotypic/karyotypic alter-
ations has been proposed [60]. hTERT is a key determinant of human telomerase enzyme
activity [100,101]. It comprises 16 exons and 15 introns and spans approximately 35 kb [102].
Life 2024, 14, 417 10 of 22

Telomerase is an enzyme responsible for maintaining telomere length [103] and was
discovered in 1985 as an enzyme capable of extending telomeric repeat sequences. How-
ever, it was not until a decade later, in 1997, that the components of the telomerase protein
complex were identified and thoroughly characterized [104]. This enzyme constitutes a
sizable ribonucleoprotein complex tasked with the synthesis of telomeric DNA repeats
in the forward direction. Broadly speaking, telomerase functions as a DNA polymerase
and is composed of two distinct subunits: a catalytic-functional component called human
telomerase reverse transcriptase (hTERT), encoded by the TERT gene, and an RNA com-
ponent known as the human telomerase RNA component (hTERC or hTR), encoded by
the TERC gene [5,102,105]. It is important to note that hTERC and hTERT are essential for
reestablishing telomerase activity [106–108].
Overall, the hTERT gene produces a 1132 amino acid polypeptide that is then converted
into a functional 130 kilodalton protein called TERT [109]. Four critical functional domains
in TERT include the N-terminal regulatory domain, the RNA-binding domain, the reverse
transcriptase domain, and the C-terminal dimerization domain [110]. Due to its complexity,
TERT is regulated at various levels, including transcriptional, post-transcriptional, and
post-translational mechanisms (Figure 4) [111–113]. Telomerase is primarily active in
proliferating cells, hematopoietic stem cells, and rapidly renewing cells [114–116].
Conversely, in somatic cells, telomerase activity is minimal or non-existent, largely due
to the tightly controlled regulation of hTERT [111]. The transfer of hTERT into human pri-
mary cells leads to an increase in telomere length and maintenance of chromosome ends. In
many cases, the forced expression of hTERT alone enables cells to suppress aging in replica-
tion and overcome the growth crisis caused by telomere shortening [117–119]. Additionally,
hTERT-immortalized cells exhibit physiological characteristics of normal cells inside the
body and maintain phenotypic markers and stable karyotypes in high passages [120,121].
Human primary cells that have been immortalized by increasing hTERT expression in
fibroblasts [122], choroidal melanocytes [31], endothelial cells [123], dermal keratinocytes,
mammary epithelial cells, osteoblasts, and pancreatic cells [124].
Some important features observed in several types of immortalized cells by increasing
hTERT expression include that they do not go towards malignancy [125,126], cell cycle
control remains normal and p53 and pRb checkpoints remain active [125,127], contact
inhibition is still normal [126], cells require growth factors for proliferation [118], and cell
karyotyping remains normal and does not show extensive changes [119,127]. The short-
ening of telomeres can only be considered an anti-cancer mechanism if cell cycle control
proteins (in checkpoint activities and telomere shortening) such as P53 and RB are working
properly. hTERT-immortalized cells combine the physiological characteristics of primary
cell lines and the continuous lifetime of cultured cell lines, preventing the aging process in
primary cell lines and the unstable karyotype of cultured cell lines. Additionally, in many
studies, hTERT-immortalized cells have been transformed into various differentiated cell
types, exhibiting tissue-specific characteristics and unique proteins and forming structures
similar to those inside the body [125].
In the field of overexpressing exogenes to immortalize cells, some genes have the ability
to immortalize a series of specific primary cells (such as the HOX family or c-Myc, which
have oncogenic activity), so using this method may affect the safety of the process. But using
the hTERT gene for immortalization can be considered a safe method for immortalizing
primary cells. A point that should not be forgotten is that hTERT is not in the category
of oncogenes [116,128].

4.3. Cancer Cells and Immortalization

Cell immortalization is a crucial stage in tumorigenesis where cells can overcome
aging and critical situations. Along this path, cells become immortal naturally and can be
isolated. Tissue samples are acquired through a biopsy, followed by a process of separation,
where various cell types are isolated and assessed for their proliferation potential and
unique characteristics. This methodology formed the foundation for obtaining cell lines,
Life 2024, 14, 417 11 of 22

commencing with the immortalized fibroblast cell line derived from mouse fibroblasts in
the 1940s. Notably, the HeLa cell line, originating from cervical cancer, represents another
significant milestone in this field [129,130].
However, in the process of the cell becoming cancerous, one of the contributing factors
is cancer-associated fibroblasts or CAFs. CAFs are the dominant stroma surrounding
the tumor. Studies have shown that CAFs can promote tumor growth, angiogenesis,
resistance to chemotherapy and metastasis, and enhance cancer progression [131,132],
thereby naturally directing cells towards immortality. A notable point in this regard is that
the primary CAFs separated from human carcinomas have been shown to remain active
even in laboratory conditions for a long period of time [133,134].
CAFs can be kept in a stable activation state to aid tumor malignancy. Epigenetic
events can affect CAFs. DNA methylation is one of the important epigenetic changes
(Figure 4). The reciprocal paracrine relationship between normal fibroblasts (NFs) and
cancer cells leads to changes in DNA methylation in NFs, which plays a central role in
NF planning for CAFs and CAF function. The presence of TGF-β1 as a primary cytokine
is also essential for CAF activation. In fact, TGF-β1 derived from cancer cells can reduce
miR-200 expression and, thus, help activate CAFs [135].
The expression of miR-200 is also concurrent with increased expression of the DNMT3B
gene in CAFs. DNMT3B is not only a direct target of miR-221 but also affects miR-200b/c
and regulates their expression in CAFs. On the other hand, miR-141 inhibition in these
cells increases the expression of transcription factor 12 (TCF12) to facilitate the growth of
breast cancer cells through CXCL12 secretion in CAFs, which leads to increased c-Myc and
cyclin D1 expression in breast cancer cells, thus allowing these cells to undergo immortality
through c-Myc activation [135].
In other words, these points indicate that the status of CAFs can be changed by
modifying epigenetic factors and thus contribute to cell immortality as an example in the
direction of proliferation. However, it is also essential to mention that cell immortalization
cannot be considered a safe and reliable method in the laboratory to promote cell malignancy
through epigenetic pathways.

4.4. Combining Methods

In some reports, the use of only one method may not be capable of producing non-
dividing cells. Therefore, depending on different cell types, it is better to combine the
cell cycle suppressor and hTERT expression to make more cells dividing. For example,
human ovarian surface epithelial cells have been made non-dividing, providing good
conditions for the study and progression of ovarian cancer. Inhibiting p53 expression with
small interfering RNA through retroviral intermediaries can delay aging and increase cell
division, but it is not enough to make natural ovarian surface epithelial cells non-dividing
and human MSCs [136,137].
Merely introducing a subunit of the catalytic telomerase is inadequate in achieving a
significant non-dividing state. However, the simultaneous suppression of p53 expression
along with the overexpression of telomerase catalytic subunits proves to be sufficient
in inducing cellular immortality in cultures of human ovarian surface epithelial cells.
Additionally, WI-38 embryonic lung fibroblasts, widely used for studies on the aging
process, have been made non-dividing with increased expression of the T antigen, hTERT,
and H-ras genes, among other cases (Figure 4) [136]. In addition, WI-38 embryonic lung
fibroblasts, which are widely used for studying the aging process, have been genetically
modified to overexpress the T antigen, hTERT, and H-ras genes [138] and many other cases.

4.5. Chemical Components and Rays Role in Immortalization

Cells can be immortalized through exposure to radioactive factors and chemical car-
cinogens. Certain chemicals, some of which are carcinogenic, have the capacity to contribute
to cell immortalization. These chemicals are categorized based on their carcinogenic poten-
tial. Each carcinogen can be further grouped based on its mode of action into genotoxic
Life 2024, 14, 417 12 of 22

carcinogens [139] or non-genotoxic carcinogens [139]. Genotoxic carcinogens are substances

or agents that directly initiate carcinogenesis by interacting directly with DNA, leading
to DNA damage and chromosomal abnormalities that can be detected through genotox-
icity testing. On the other hand, NGCs are agents capable of inducing cancer through a
secondary mechanism, often as a result of their indirect impact on DNA. They have the
ability to alter signal transduction pathways or gene expression. The GCs can be detected
using genotoxicity testing, which detects changes to the cell at the molecular and cellular
levels. These changes include mutations in genes, DNA strand breaks, the formation of
DNA adducts, chromosomal aberrations, and aneuploidy [140]. As an illustration, potent
mutagenic carcinogens like N-methyl-N-nitrosourea (MNU) and benzo(a)pyrene [141] have
been demonstrated to be effective agents for immortalization in Syrian hamster dermal
cells [142] cell transformation assay [143] through the direct inactivation of the tumor
suppressors p53 and p16 (Figure 4) [144].
Physical carcinogens (such as ionizing radiation) are also powerful immortalization
agents with different mechanisms and frequencies in rodent and human cells [140]. For
example, X-rays, neutrons, and gamma rays produce immortal clones in SHD cells [144].
Conversely, the immortalization of human mammary cells through ionizing radiation is
a relatively rare occurrence [145]. Similarly, methyl sulfate, a powerful clastogen, is an
efficient immortalizing carcinogen in mammalian SDH cells and Chinese hamster cells [143]
and has a similar mode of action to that of ionizing radiation.
Furthermore, the carcinogenic strength of basic aliphatic alkylating agents like alkylni-
trosamides and alkylmethanesulfonates is directly associated with their capacity to modify
the comparatively less reactive oxygen atoms in DNA, notably the O6 atom of guanine [143].
In addition there are data suggesting that acetaminophen activates telomerase [146–149],
which could lead to the immortalization of cells. However, there are also data indicating
that acetaminophen can inhibit CDK4 and CDK2, thus imposing a cell cycle checkpoint at
G1 and effectively blocking cellular proliferation [140].
There are other data that show that aspirin, at therapeutically relevant concentrations,
prevents the senescence of endothelial cells. This effect seems to be due to increased nitric
oxide (NO) synthesis and reduced oxidative stress. Consistent with these findings, the
formation of asymmetric dimethylarginine [150], an endogenous inhibitor of NO synthase,
was decreased and the activity of dimethylarginine dimethylaminohydrolase (DDAH), an
enzyme that degrades ADMA, was decreased [149].

4.6. Gene Manipulation and Immortalization

The CRISPR/Cas9 genome-editing system provides us an unprecedented opportunity to
target and modify genomic sequences with high levels of efficacy and specificity [151–155].
The CRISPR/Cas9 system induces DNA double-strand breaks at specific sites of genomic
DNA, which should allow a safer and more targeted gene delivery of the immortalizing
genes [156]. Indeed, the most potent gene editing tool to date is encapsulated by the Clustered
Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein
(CRISPR/Cas9) system [157].
This can be considered one of the most powerful and versatile forms of technology
both for gene editing and transcriptional control but also for epigenetic modulation [158].
For example, in order to overcome the technical challenge of maintaining primary BMSCs
in long-term culture, mouse bone marrow stromal stem cells (BMSCs), which are one of
the most common mesenchymal stem cells, have been reversibly immortalized mediated
CRISPR/Cas9, which can maintain the multipotent characteristics of mesenchymal stem
cells (MSCs). In this study, by exploiting the CRISPR/Cas9-based homology-directed-repair
(HDR) mechanism, they targeted SV40T to mouse Rosa26 locus and efficiently immortalized
mouse BMSCs (i.e., imBMSCs) (Figure 4) [156].
Certainly, the study demonstrates that CRISPR-Cas9-mediated targeting of the p53
gene or CDKN2A locus proves to be an effective method for immortalizing primary mar-
moset skin fibroblasts. This is particularly significant as the common marmoset serves as a
Life 2024, 14, 417 13 of 22

valuable non-human primate model for studying various human diseases. The research
reveals that, similar to Cdkn2a-deficient mouse cells, CDKN2A-deficient marmoset cells
express wild-type p53 proteins and respond normally to genotoxic stresses such as adri-
amycin and etoposide. These findings collectively underscore that Cas9-mediated gene
targeting of the p53 gene or CDKN2A locus is a potent tool for establishing immortalized
marmoset cell lines with specific genetic modifications [159].

5. Challenges of Using Cell Lines

When opting for cell lines over primary cells, it is crucial to bear certain considerations
in mind. Because cell lines undergo genetic manipulation, their phenotype, inherent func-
tions, and responses to stimuli may undergo alterations. Another critical aspect to consider
is the repeated cultivation of cell lines, which can bring about shifts in genotypic and phe-
notypic features over prolonged periods. This may result in genetic drift and heterogeneity
within cultures and, consequently, may lead to variations in results. Consequently, cell
lines may not entirely mirror all the attributes of primary cells in the long term and may
yield differing outcomes. Two significant concerns related to cell lines are contamination
by other cell lines and mycoplasma. Mycoplasma contamination, in particular, can persist
undetected in cell cultures for extended periods, resulting in substantial alterations in gene
expression and cellular behavior. This underscores the importance of vigilant monitoring
and maintenance practices when working with cell lines [160,161]. Also, a 2011 study of
122 different head and neck cancer cell lines revealed that 37 (30%) were misidentified [162].
Analyses of a variety of tissue culture collections and cells sent to repositories for curation
and storage from labs in the United States, Europe, and Asia suggest that at least 15% of
cell lines are misidentified or contaminated [162,163].
In addition, a number of factors contribute to the problems of cell line misidentification
and contamination. For example, inadvertently using a pipette more than once when work-
ing with different cell lines in culture can lead to cross-contamination. If the contaminating
cell line divides more rapidly than the original cells, it can quickly dominate the population,
changing the identity of the culture. This event often goes undetected because cells from
different sources can be morphologically similar [164]. Walter Nelson-Rees brought to light
a harsh reality in the early 1970s regarding the widespread cross-contamination of cell lines,
both between different species and within the same species. He demonstrated that during
that period, a significant portion of cell lines utilized globally and distributed by cell banks
were tainted with HeLa cells. This revelation had profound implications for cell-based
research and emphasized the need for rigorous quality control measures (Table 1) [165].

Table 1. Types of immortalized cells along with some of their acquired characteristics.

The
Immortal The Method Gene Transfer Immortal Cell
Strategy Effectiveness Immortality Result Ref.
Cell Type of Immortality Method Characteristics
of the Method

They maintained - The first generation of

their phenotypes immortalized hVFE
with almost cells obtained from
identical genotypes the luminal surface of
in cellular the true VF.
pathways and - The immortalized
Expression of functioned properly hVFE can be easily
Human Increased Most effective frozen, stored,
the catalytic in relation to ion
1 vocal fold hTERT gene and safe in Retrovirus and recovered. [30]
subunit of and protein
epithelial cells expression most cell types - It can be shipped to
telomerase transport and cell
signaling. They other research
also maintained the institutions and
ability of stable widely used for
reproduction for various in vitro
more than research in
8 months. laryngeal field.
Life 2024, 14, 417 14 of 22

Table 1. Cont.

The
Immortal The Method Gene Transfer Immortal Cell
Strategy Effectiveness Immortality Result Ref.
Cell Type of Immortality Method Characteristics
of the Method
They maintained
their biological
characteristics and - The CCEC-SV40T line
had a stronger was successfully
Induction
Usually proliferation established.
Primary canine of viral
effective but capacity than - Can be used for
2 corneal SV40 T antigen oncogenes that Lentivirus [166]
not safe normal cells. They in vitro studies, such
epithelial cells inactivate cell
(viral gene) also maintained as research on corneal
cycle proteins
their diploid diseases or
karyotype drug screening.
and serum-
dependent ability.
They maintained
the morphological - The immortalized
and functional cell line
Expression of characteristics of SV40T-YREC-hTERT
the catalytic A combination the primary cells was established for
subunit of of methods is and also had the first time.
Increased
telomerase + usually functions related to - SV40T-YREC-hTERT
Yak rumen hTERT gene
3 Induction effective, but Lentivirus the normal provided an [167]
epithelial cells expression +
of viral there is caution transport and experimental model
SV40 T antigen
oncogenes that in using absorption of for studying the
inactivate cell viral genes short-chain fatty biological function of
cycle proteins acids. Cell the yak rumen
proliferation and epithelium in vitro.
karyotype
were normal.

- The hTERT RPE-1

Life expectancy ERT2-Cre-ERT2 cell
increased. It does line was established.
Human retinal Expression of ERT2-Cre- - Is versatile in gene
Increased Most effective not have
pigment the catalytic ERT2AAVS1 editing and can
4 hTERT gene and safe in transformed [31]
epithelial subunit of integration facilitate future
expression most cell types phenotypes and has
RPE-1 cells telomerase plasmid functional studies of
a stable and normal
karyotype. genes and the
human genome.

Cells showed
longer lifespan and
maintained
endothelial
- This study is the first to
Expression of characteristics.
Increased Most effective confirm the antitumor
the catalytic They expressed the
5 HUVECs hTERT gene and safe in Lentivirus immunity of hTERT- [168]
subunit of factors CD31,
expression most cell types immortalized
telomerase VEGFR-II, and
HUVECs.
alpha5 integrin.
Antitumor
immunity was
also confirmed.

- Telomerized hMSC
lines maintain
The restoration of long-term
telomerase activity, self-renewal and
the increase in the differentiation
life span of the cells, capacity.
Human bone Expression of the characteristics - hMSC-TERT cell lines
Increased Most effective were capable of
marrow the catalytic of the stem cells of
6 hTERT gene and safe in Retrovirus forming bone, [169]
mesenchymal subunit of self-renewal, and
expression most cell types bone-marrow
stem cells telomerase the ability to
differentiate into supporting stroma,
the mesoderm-type and adipocytes when
cell lineage transplanted
were preserved. subcutaneously in
immune-
deficient mice.
Expression of
A combination A stable cell line
the catalytic - A conditional human
Increased of methods is was obtained from
subunit of fetal hepatocytes
hTERT gene usually human fetal liver
Human fetal telomerase + Vector + E. coli cell line with
7 expression + effective, but cells that was able [170]
hepatocytes overexpres- plasmid mesenchymal
SV40 T there is caution to secrete
sion of certain characteristics
antigen + E7 in using albumin–urea and
genes for im- was established.
viral genes consume glucose.
mortalization
Life 2024, 14, 417 15 of 22

Table 1. Cont.

The
Immortal The Method Gene Transfer Immortal Cell
Strategy Effectiveness Immortality Result Ref.
Cell Type of Immortality Method Characteristics
of the Method
It produces stem
cells with increased
proliferation - This line having
capacity and they been established
Human neural Expression of using a much lesser
8 Retrovirus do not change [95]
stem cells the v-myc gene mutated and better
shape in laboratory
conditions and are characterized
not tumorigenic in c-myc mutant.
the body.

- SV40-transfected
ligamentocytes
express normal
tendon components.
- However, some
differences, such as
Ligamentocytes Transfected distinct cytoskeletal
Induction
derived from Usually ligamentocytes changes and limited
of viral
human effective but Vector maintain the survival in long-term
9 SV40 T antigen oncogenes that [171]
anterior not safe transfection phenotype and 3D cultures, could be
inactivate cell
cruciate (viral gene) vital properties of demonstrated.
cycle proteins
ligament the cell. - It follows that tissue
engineering (TE)
with transfected SV40
cells is only possible
for limited
culture periods.

- An hTERT-
Proliferated longer BMSCs/Tet-on/GAL
than wild-type cell line was
BMSCs. Phenotype constructed
and karyotype were using a single
not significantly Tet-on-inducible
different from lentivirus system.
non-transfected - Subsequent
Expression of
Bone marrow Increased Most effective cells. The cells also experiments
the catalytic
10 mesenchymal hTERT gene and safe in Lentivirus maintained the demonstrated that the [156]
subunit of
stromal cells expression most cell types normal secretion of rat GAL
telomerase
morphology and from hTERT-
neural BMSCs/Tet-on/GAL
differentiation was switched on and
characteristics of off under the control
stem cells when of an inducer in a
cultured in dose-dependent
induction media. manner.

It is important to remember that cell lines may not exhibit the same behavior as
primary cells. To bolster the validity of these results, it is essential to consistently conduct
crucial control experiments using primary cells [172]. However, the potential challenge
lies in the time and costs involved in conducting these tests. Nevertheless, based on the
explanation provided, it is imperative to exercise caution when working with cell lines.
It is strongly recommended to include experiments that validate key findings in primary
cultures and to meticulously adhere to all relevant considerations.

6. Conclusions
Cell immortalization proves to be a valuable technique for acquiring cell lines with
unrestricted replicative capacity. By following this approach, cells can surpass the Hayflick
limit and evade the mechanisms linked to replicative senescence and eventual cell apoptosis.
This is achieved through the upregulation of various viral oncogenes/oncoproteins, the
TERT component of telomerase, and the expression of genes that regulate the cell cycle,
ultimately leading to conditional immortality [61]. In addition, the use of immortal cell
lines has been helpful in many researches, some of which were mentioned in this article,
and these cases can present a sign of the importance of creating a cell line through the
immortalization technique. It is worth noting that along with all the advantages that cell
immortality provides for research, there are still problems (such as manipulation of the cell
Life 2024, 14, 417 16 of 22

cycle when p53 and Rb change, chromosomal instability, etc.), and their solution requires
more research in the future.

Author Contributions: Conceptualization, M.C., M.H. and S.R.; writing—original draft preparation.
M.C., M.H. and S.N.M.; editing, M.Y., S.R., S.A. and F.H.; visualization, M.C.; supervision, M.H.;
project administration, S.R. and F.H.; funding acquisition, F.H. All authors have read and agreed to
the published version of the manuscript.
Funding: This research was funded by the University of Adelaide, research consultation account
(found for publication).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflicts of interest.

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